Characterisation of a new molecule based on two E2 sequences from bovine viral diarrhoea-mucosal disease virus fused to the human immunoglobulin Fc fragment

Figures

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  Fig 1. Expression of the bvde2fc gene and assembly of the recombinant adenoviral vector carrying the gene (AdBVDE2Fc) A – Scheme of the transcriptional unit for bvde2fc gene expression. pCMV – early/immediate cytomegalovirus promoter; S – spacer of glycine and serine; H – histidine tag; hFc – Fc segment of human immunoglobulin; bGH poly (A) – sequence of the transcriptional termination from the bovine growth hormone B – Expression assay of the bvde2fc gene by transfecting the pcDNA 3.1- bvde2fc plasmid into HEK-293 cells. MWM – molecular weight marker; 1 – supernatant of HEK-293 cells transduced without plasmid; 2 – supernatant of HEK-293 cells transduced with the pcDNA 3.1- bvde2fc plasmid; WB – Western blot C – Fluorescent halo indicating the correct assembly of AdBVDE2Fc

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  Fig 2. Expression of the bvde2fc gene by transducing mammalian cell lines from different species with AdBVDE2Fc. 1 – supernatant of non-transduced cells; 2 – supernatant of transduced cells with AdBVDE2Fc at MOI 100

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  Fig 3. Purification of BVDE2Fc by IMAC A – Analysis by SDS-PAGE and Western blot of different steps of the purification process. 1 – initial sample; 2 – unbound material; 3 – wash at 20 mM imidazole; 4 – elution at 80 mM imidazole B – Analysis by SDS-PAGE and Western blot of the purified BVDE2Fc. 1 – in reduced conditions; 2 – in non-reduced conditions C – HPLC size exclusion chromatography of the purified BVDE2Fc. Upper graph – external standard. 1 – 660 kDa; 2 – 440 kDa; 3 – 158 kDa; 4 – 75 kDa; 5 – 43 kDa; 6 – 13.7 kDa. Lower graph – BVDE2Fc. T – tetramers; D – dimers; M – monomers

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  Fig 4. Analysis of the N-glycan profile released after BVDE2Fc deglycosylation A – N-glycan analysis by a NP-HPLC-Amide 80 column. The N-glycan retention times were expressed as glucose units (GU) by comparison with a standard 2AB-labeled dextran hydrolysate B – N-glycan analysis by HPLC-weak anion exchange chromatography. Blue – N-glycans of IgG as external standard; Red – N-glycans of BVDE2Fc; RT – retention time Inset – SDS-PAGE of BVDE2Fc. 1 – glycosylated with PNGase-F; 2 – deglycosylated with PNGase-F

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  Fig 5. Characterisation of BVDE2Fc as immunogen A – Humoral immune response after inoculating mice with 20 μg of BVDE2Fc B – Analysis of mice sera by coating with deglycosylated BVDE2Fc in ELISA assays C – Analysis of mice sera by coating with EPOFc in ELISA assays. NC – negative control; NC t:42 – negative control of the pool from mice sera at 42 days. The arrow indicates the second inoculation. Bars represent the standard deviation. OD values were compared by the Kruskal–Wallis test and the Dunn post-test (A, B), and by the Mann–Whitney test (C). * P < 0.05; ** P < 0.01; *** P < 0.001

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  Fig 6. Evaluation of sera from bovines positive to different BVD-MDV subtypes by coating with BVDE2Fc in ELISA assays. Bars represent the standard deviation. OD values were compared by the Kruskal–Wallis test and the Dunn post-test (A, B), and by the Mann–Whitney test (C). * – P < 0.05; ** – P < 0.01; *** – P < 0.001; AI – animal identification; VS – viral subtype; NC – negative control; ND – not determined; black line – cut-off value